Dr. R. JAYABALAN Assistant Professor Food and Bioprocess Technology - - PowerPoint PPT Presentation

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SCREENING FOR B I O G E N I C A M I N E P R O D U C T I O N B Y L A C TO B A C I L U S S P E C I E S A N D D E V E L O P M E N T O F F U N C T I O N A L F O O D , T E A C U R D . Dr. R. JAYABALAN Assistant Professor Food and Bioprocess

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Dr. R. JAYABALAN

Assistant Professor Food and Bioprocess Technology Laboratory Department of Life Science National Institute of Technology, Rourkela Odisha -769008, INDIA.

SCREENING FOR B I O G E N I C A M I N E P R O D U C T I O N B Y L A C TO B A C I L U S S P E C I E S A N D D E V E L O P M E N T O F F U N C T I O N A L F O O D , T E A C U R D .

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INTRODUCTION

Tea – most popular beverage in the world next

to water

Antioxidants – tea polyphenols – tea catechins
Reduction of cholesterol, protection against

cardio-vascular disease, and cancer

Curd – Indian Yoghurt, home made, inoculum

from previous curd

Daily food
Tea curd – functional food – tea polyphenols –

biologically active compounds – health benefits

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INTRODUCTION

Biogenic amines - organic, basic nitrogenous

compounds with one or more amine groups

Removal of carboxyl group from amino acids

by amino acid decarboxylase enzyme

Alkaline in nature

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(HI) (TY) (TR) (PUT) (CAD)

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INTRODUCTION

Excessive levels – Hypotension, hypertension,

nausea, respiratory distress, heart palpitation

Histamine – vaso active effects
Tyramine – rise in blood pressure, mutagen

precurson

Putrescine, cadaverine : can be converted into

nitrosamines – potential carcinogens

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OBJECTIVES

To isolate probiotic bacteria from locally

available curd (OMFED, Rourkela, Odisha, India and home made)

To characterize the bacteria for biogenic

amine production and tolerance to acidic pH

To utilize the non biogenic amine producing

bacteria for the development of green tea and black tea curd

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METHODS

ISOLATION OF PROBIOTIC MICROBES
OMFED curd, Rourkela, Odisha, India and home made
Handia – home made rice fermented with inoculum from previously fermented –
btained from local seller
MRS agar, standardized procedure
37°C for 24 hours
Morphological identification – simple staining
Grown cultures – repeatedly subcultured for 5 times in MRS broth
SCREENIGN FOR BIOGENIC AMINE PRODUCTION
Decarboxylase media with different amino acids
Wells – 0.5 cm in agar plate
250 µl cultures in MRS broth – inoculated
ACID TOLERANCE TEST
MRS broth cultures – centrifuged at 10,000 rpm for 5 minutes – pellet –

resuspended in PBS buffer with pH 7.4, 5.5, 4.0, 3.0 and 2.0

24 hours incubation at 37°C
Plated on MRS agar to check viability

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Components ( grams / litre) Proteose peptone 10.000 Beef extract 10.000 Yeast extract 5.000 Dextrose 20.000 Polysorbate 80 1.000 Ammonium citrate 2.000 Sodium acetate 5.000 Magnesium sulphate 0.100 Manganese sulphate 0.050 Dipotassium phosphate 2.000 Agar 15.000 pH 6.5±0.2

Components

( grams / litre)

Tryptone 5.0 Yeast extract 5.0 Meat extract 5.0 Sodium chloride 2.5 Glucose 0.5 Tween 80 1.0 Magnesium sulphate 0.2 Manganese sulphate 0.05 Ferrous sulphate 0.004 Ammonium citrate 2.0 Thiamine 0.001 Di-Potassium phosphate 2.0 Calcium carbonate 0.10 Pryridoxal-5- phosphate 0.05 Amino acid* 1.0 Bromocresol purple 0.06 Agar 20 pH 5.3

Composition of decaboxylase media Composition of MRS agar

*Control media lacks amino acid. Amino acids ornithine, tyrosine, lysine and histidine added separately

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METHODS

PREPARATION OF TEA INFUSIONS
Green tea and Black tea – Camellia sinensis (L) O. Kuntze – Parry Agro

Industries Ltd., Valparai, Tamil Nadu, India

2% (strength of normal cup of tea) – boiled water – 5 minutes – infusion
Curd manufacture – tea infusions in milk
HPLC analysis – tea infusions in water

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PREPARATION OF TEA CURD (Jaziri, 2009) Milk – OMFED (toned, 3.0% fat and standardized 8.5% SNF), Rourkela, Odisha, India 2% green tea and black tea, separately 5 minutes with stirring Filtered through sterile cotton Cooled to 45°C Lactobacillus cultures (grown in MRS broth, 3 X 106) – 3.3 mL of each culture (10% v/v inoculation) Sterile, tightly capped tubes (30 mL) Inoculation – 6 hours at 42°C Stored at 4°C in a refrigerator

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SAMPLING

Periodic sampling
Each tube – only once to avoid contamination
After 6 hours – considered as 0 day
Kept in refrigerator
Sampling done at the end of 1, 7, 14, and 21 days

MICROBIOLOGICAL ANALYSIS

Aseptic removal of sample
serial dilution in 0.1 % peptone water
Standard spread plate technique using MRS agar
37°C incubation for 48 hours under aerobic conditions.

DETERMINATION OF pH

Electronic pH meter (Orion model 290A)

DETERMINATION OF TITRATABLE ACIDITY

10 g sample – titrated against 0.1 N NaOH
% of lactic acid = ml of alkali× normality of NaOH×9/weight of sample (g)

HPLC ANALYSIS OF TEA POLYPHENOLS

Tea infusion prepared in water and inoculated with microbial cultures

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HPLC ANALYSIS OF TEA POLYPHENOLS (Anon, 1999)

5 mL of sample (tea infusion in water) – extracted with 20 mL methanol Filtered through 0.45 µM membrane filter 10 µl of filtrate Shimadzu (Kyoto, Japan) – HPLC system with PDA (SPD-M10Avp) Phenomenex Luna C-18(2) column (4.6 mm ID X 25 cm, 5 uM) Mobile phase: Mixture of 0.1% orthophosphoric acid (A) and acetonitrile (B) Gradient elution: 0-12 min, 15% B ; 12-22 min, 25%, 22-30 min, 15% B) Flow rate – 1.0 mL/min, 35°C Detection: 280 nm Resolution peaks recorded – accodring to retention time of compound Standard curves – quantification of tea polyphenols

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Figure 1: Morphology of bacteria isolated from OMFED curd and home made curd OF1 OF2 OF4

RESULTS AND DISCUSSION

HM1 HM2

Number Morphology Bacteria / Yeast 1.OF1 Long rod Bacteria 2.OF2 Oval shape Yeast 3.OF3 Long rod Bacteria 4.OF4 Long rod Bacteria 5.HM1 Long rod Bacteria 6.HM2 Long rod Bacteria 7.HN1 Oval shape Yeast 8.HN2 Oval shape Yeast

Table 1: Morphology of bacteria isolated from OMFED curd, home made curd and Handia

ISOLATION OF PROBIOTIC BACTERIA

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SCREENING FOR BIOGENIC AMINE PRODUCTION

Figure 2: Screening for biogenic amine production in decarboxylase media control lacks amino acid

1.OF1 2.OF2 3.OF3 4.OF4 5.HM1 6.HM2 7.HN1 8.HN2

Figure 3: Screening for biogenic amine production in decarboxylase media with tyrosine Tyramine production Positive : OF1, OF2, HM1, HN1 Negative: OF3, OF4, HM2, HN2 Control media : color production due to amino acids in proteins

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1.OF1 2.OF2 3.OF3 4.OF4 5.HM1 6.HM2 7.HN1 8.HN2

Figure 4: Screening for biogenic amine production in decarboxylase media with

rnithine

Putrescine production Positive : OF1, HM1 Negative: OF2, OF3, OF4, HM2, HN1, HN2 Figure 5: Screening for biogenic amine production in decarboxylase media with lysine Cadaverine production Positive : HM1 Negative: OF1, OF2, OF3, OF4, HM2, HN1, HN2

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Figure 6: Screening for biogenic amine production in decarboxylase media with histidine

1.OF1 2.OF2 3.OF3 4.OF4 5.HM1 6.HM2 7.HN1 8.HN2

Histamine production Positive : OF1, HM1 Negative: OF2, OF3, OF4, HM2, HN1, HN2

Table 2: Summary of screening for biogenic amine production

Tyrosine (Tyramine) Ornithine (Putrescine) Lysine (Cadeverine) Histidine (Histamine) Selection OF1 Yes Yes No Yes No OF2 Yes No No No OF3 No No No No Yes OF4 No No No No Yes HM1 Yes Yes Yes Yes No HM2 No No No No Yes HN1 Yes No No No No HN2 No No No No

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Acid tolerance test

Bacteria pH 7.4 pH 5.5 pH 4 pH 3 pH 2 OF3 GROWTH GROWTH GROWTH GROWTH NO GROWTH OF4 GROWTH GROWTH GROWTH GROWTH NO GROWTH HM2 GROWTH GROWTH GROWTH GROWTH NO GROWTH

Table 3: Effect of pH on growth of selected non-biogenic amine producing probiotic bacteria

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Green tea curd Black tea curd Figure 7: Green tea curd and black tea curd

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Refrigerated storage (day) pH Titratable acidity (gram lactic acid / liter) Number of bacteria (CFU/mL) 4.10 1.26 2.27 X 106 1 4.04 1.35 1.84 X 106 7 4.00 1.71 1.36 X 103 14 3.80 1.17 1.11 X 103 21 4.10 1.62 contamination

Table 4: Effect of time on pH, titratable acidity and number of bacteria of green tea curd

Reduction of bacterial number after 7 days. Study required after 1 day.

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Time Refrigerated storage (day) pH Titratable acidity (gram lactic acid / liter) Number of bacteria (CFU/mL) 4.10 1.35 2.30 X 106 1 4.00 1.44 2.00 X 106 7 3.60 1.80 1.61 X 103 14 3.50 1.26 contamination 21 4.05 1.53 contamination

Table 5: Effect of time on pH, titratable acidity and number of bacteria of black tea curd

Reduction of bacterial number after 7 days. Study required after 1 day.

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Figure 8: Liquid Chromatogram of tea polyphenols

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Refrigerated storage (day) EGCG (mg /L) EGC (mg /L) ECG (mg /L) EC (mg /L) Catechin (mg /L) Gallic acid (mg /L) Caffeine (mg /L) 65.2 37.9 11.1 8.5 1.1 5.2 27.1 1 16.7 16.0 2.9 3.7 0.7 2.7 13.9 7 20.9 51.6 2.9 14.8 0.6 7.7 52.1 14 13.3 45.6 0.3 0.1 0.3 6.9 36.5 21 1.5 4.6 0.4 15.7 0.2 2.2 39.5

Table 6: Effect of refrigerated storage on content of tea polyphenols in green tea curd

Varying stability. Conversion / degradation of complex molecules into simpler molecules Tea polyphenols are stable at acidic pH. Hence, microbial enzymes may be the reason for observed varying stability

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Refrigerated storage (day) EGCG (mg /L) EGC (mg /L) ECG (mg /L) EC (mg /L) Catechin (mg /L) Gallic acid (mg /L) Caffeine (mg /L) 5.3 1.3 1.7 0.1 0.9 2.9 39.2 1 1.5 1.4 0.4 ND 0.4 0.5 16.9 7 5.1 0.1 0.6 0.3 0.3 5.4 73.9 14 3.4 0.1 0.2 ND ND 3.7 65.4 21 3.4 0.8 0.6 0.2 0.5 4.6 64.2

Table 7: Effect of refrigerated storage on content of tea polyphenols in black tea curd

ND – Not Detected Varying stability. Conversion / degradation of complex molecules into simpler molecules Tea polyphenols are stable at acidic pH. Hence, microbial enzymes may be the reason for observed varying stability

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Among 8 microorganisms isolated, 3 were selected to produce tea curd (non-biogenic amine producing bacteria) Tolerant up to pH 3.0 and not at pH 2.0 Green tea curd and black tea curd were prepared Refrigerated storage – reduction of bacterial number after 7 days. Tea polyphenols – varying stability Concentration of tea polyphenols after 1 day – can be

Sweet green tea and black tea curd
Frozen green tea and black tea curd (without liquid)

CONCLUSION

FUTURE WORKS

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NIT Rourkela Department of Science and Technology, Government of India Parry Agro Industries Ltd., Valparai, Tamil Nadu, India Research team

Ms. Indira Dash
Ms. Moumita Sahoo,
Mr. Ajay Dethose,
Ms. Banishree Sahoo

ACKNOWLEDGEMENTS

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References

Jaziri, I., Slama, M.B., Mhadhbi, H., Urdaci, M.C., Hamdi, M. 2009. Effect of

green and black teas (Camellia sinensis L.) on the characteristic microflora on yogurt during fermentation and refrigerated storage. Food Chemistry, 112: 614- 620

Shalaby, A. R. (1996). Significance of biogenic amines to food safety and

human health. Food Research International, 29, 675–690.

Moinard, C., Cynober, L., & de Bandt, J. P. (2005). Polyamines: Metabolism

and implications in human diseases. Clinical Nutrition, 24, 184–197.

Silla Santos, M. H. (1996). Biogenic amines: their importance in foods.

International Journal Food Microbiology, 29, 213–231.

Brink, B. T., Damink, C., Joosten, H., & Huis isn't Veld, J. (1990). Occurrence

and formation of biologically active amines in foods. International Journal of Food Microbiology, 11, 73–84.

Bjeldanes, L. F., Schutz, D. E., & Morris, M. M. (1978). On the etiology of

scombroid poisoning: cadaverine potentiation of histamine toxicity in the guinea-pig. Food and Cosmetics Toxicology, 16, 157–159.

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